A Biography of Sherman J. Silber’s Research in Reproductive Medicine

Beginning with his first papers on kidney transplantation in inbred rats in 1973, Dr. Silber developed the whole field of microsurgery of the urogenital and reproductive systems. He first developed these microsurgical techniques with the goal of using them in rats and mice that were sufficiently inbred to avoid immune rejection. With this technique he elucidated the physiology of compensatory and obligatory hypertrophy of the kidney after unilateral nephrectomy. He then utilized these techniques to develop antigen-specific methods of immunosuppression for organ transplantation. These early papers were published in the American Journal of Physiology, the American Journal of Surgery, JAMA, the Journal of Urology, theAustralian and New Zealand Journal of Surgery, and in Transplantation.

In 1976, he applied these techniques in the human to successfully autotransplant abdominal testes into the scrotum in children with otherwise uncorrectable cryptorchidism. In 1977, he expanded from testis autotransplantation to homografting testes to anorchic men from their identical twin brothers. These papers appeared in Journal of Andrology and in Fertility and Sterility in the late 1970’s.

2. Application of Microsurgery to Vasectomy Reversal and Vasoepididymostomy for Obstructive Azoospermia

He first published the two-layer technique for vasovasostomy for reversal of vasectomy in Surgery, Gynecology and Obstetrics in 1976. This was a clinical culmination of six years of microsurgical research that began in 1970 in the animal lab at the University of Michigan. He subsequently published follow-up papers on microscopic vasectomy reversal between 1976 and 1978 in Fertility and Sterility, Urology, Lancet, and Urologic Clinics of North America.

There were several unexpected findings from this research which have allowed vasectomy reversal today to be well over 90% successful compared to 1975 when there were only occasional and unpredictable successes with vasectomy reversal. Among those findings were: 1) If there are no sperm in the vas fluid at the time of vasovasostomy, the operation will not be successful no matter how perfectly the anastomosis was performed. The cause of this failure was found to be epididymal blockage secondary to either extravasation or concretions in the epididymis caused by long-term pressure build-up after vasectomy; 2) Next, he developed the technique for specific anastomosis of the vas lumen to the epididymal tubule proximal to site of secondary blockage. This had never been done before. Up until that time all vasoepididymostomies had been performed in a crude fashion that only encouraged the formation of a fistula by cutting through the epididymis through many sections of the tubule and anastomosing the vas to the outer epididymal tunic, hoping the fistula would form and result in patency. However, the technique he developed involved the specific anastomosis of the inner lumen of the vas, mucosa to mucosa, to the epididymal tubule, and then an outer layer suturing of the vas muscularis to the outer epididymal tunic for support. This methodology raised the success rate for vasoepididymostomy from 10% to 93% currently. Furthermore, it explained the reason for the frequent failure of vasectomy reversal even when a competent microsurgeon was performing the vas-to-vas anastomosis; 3) Finally, in 1977, he published in Lancet the first association of sperm granuloma (resulting from leakage at the vasectomy site), with success of simple vasovasostomy. He then introduced an “open-ended” technique which involves the secure, safe sealing of the abdominal end of the vas deferens to avoid vasectomy failure, but leaving the testicular end open so that a sperm granuloma will intentionally form, thus avoiding secondary epididymal obstruction, which occurs if there is no leakage at the vasectomy site.

3. Testicle Homotransplantation

In 1978, he published in Fertility and Sterility the first successful case of transplantation of a human testicle for anorchia. Subsequently, the recipient had a normal sperm count and fathered five children. Furthermore, rather than having to go on testosterone replacement which was very difficult at that time, the recipient required no medication, and has normal and constant testosterone levels. He utilized this unique case to study the physiology of transplant ischemia in the testes and the recovery time of normal spermatogenesis. He followed up patients who received autotransplantation for cryptorchidism in the 1980’s and was the first to report that it was possible for recovery of normal spermatogenesis in these patients even if the autotransplant was performed as a young adult.

After the initial reports by Winston in England and Gomel in Canada, he performed the first microsurgical fallopian tube reversal in the United States, and was the first to report in a documented fashion that the prediction of pregnancy was dependent upon the length of the fallopian tube remaining after the reanastomosis. The technique is performed through a small mini-laparotomy with an operating microscopic as a simple, one-day stay procedure, and most women, if given the choice, still prefer this to IVF.

5. Quantitative Analysis of Testicle Biopsy

In 1981, he published with Dr. Rodriguez-Rigau results with attempting to predict sperm count based upon a quantitative analysis of spermatogenesis on testicle biopsy histology. At that time, in 1981, he was able to predict sperm count after surgery for vasectomy reversal or other obstruction based on a count of the number of mature spermatids (or sperm) seen within the testes biopsy per seminiferous tubule. What was not realized in 1981, is that in 1994, thirteen years later, this would eventually lead to the technique developed by he and Paul Devroey of TESE (testicular sperm extraction) for cases of non-obstructive azoospermia.

What he discovered, back in 1981, had its clinical benefit come to fruition thirteen years later with TESE-ICSI. Azoospermic men, despite what seemed to be no sperm production at all, in 60% of cases, had some tiny amount of spermatogenesis that could be found somewhere in one of the seminiferous tubules on testes biopsy specimen. Despite apparent absence of spermatogenesis throughout the testis, with the advent of ICSI, this led to the possibility of pregnancy for the wives of men with non-obstructive azoospermia.

6. Transurethral Resection for Ejaculatory Duct Obstruction

In 1980, he published the first paper on “transurethral resection for treating ejaculatory duct obstruction,” a rare condition, but one that heretofore had been considered untreatable. This has now become a standard procedure for men whose ultrasound shows ejaculatory duct blockage.

7. Laparoscopy in Urology

In 1980 he performed the first laparoscopy for urologic purposes in the male entitled “Laparoscopy for cryptorchidism,” in theJournal of Urology. He was the first to recommend this technique for localization of nonpalpable testes prior to contemplated surgical correction.

8. Microsurgical Varicocoelectomy

He has incurred a great deal of enmity from some urologic colleagues because of his position on varicocoele (that is, he demonstrated that varicocoelectomy is not at all beneficial in the treatment of the infertile couple). He was, nonetheless, the first to recommend a microsurgical approach so as to avoid the potentially damaging side effects of varicocoelectomy, which would include devascularization in occasional cases and, more commonly, post-operative hydrocoele. By avoiding ligation of the lymphatics, or of the arteries, and only specifically ligating the veins under an operating microscope, he was the first to maintain that varicocoelectomy could be performed more safely with microsurgical technique (Fertility and Sterility, 1979).

In 1987, he reported in Lancet the first successful case of pregnancy with congenital absence of the vas and microsurgical sperm retrieval (MESA) using IVF. He followed this up with a detailed report in the New England Journal of Medicine in 1990, even before ICSI, demonstrating the possibility of pregnancy from sperm retrieved from the epididymis and utilized for IVF. He was the first to demonstrate that in the obstructed state, sperm from the distal epididymis are the least motile and the least effective for pregnancy, and sperm from the proximal epididymis, quite contrary to what one might expect, had the greatest motility and the best chance for success with IVF. This was because the more proximal sperm were more recently produced and thus had not undergone senescence yet as had the distal epididymal sperm. This is the exact inverse of what one would expect in the normal, non-obstructed epididymis.

In 1993, fairly shortly following the paper of Anguiano et al., he published almost simultaneously with Anguiano, the association of the mutations in the cystic fibrosis gene with congenital absence of the vas deferens. Until this time, there had been no understanding of the etiology of this condition (CBVAD). Furthermore, unlike the Anguiano study, he followed the CF mutations through three generations, from the father of the patient, the patient, and to the male and female offspring of the CF patient.

In 1993, the collaboration began of Dr. Silber with Dr. Paul Devroey and Dr. Andre Van Steirteghem of Belgium to see whether success rates with sperm retrieval for obstructive azoospermia (such as congenital absence of the vas), could be increased using ICSI. He demonstrated that the pregnancy rate with ICSI using microsurgically retrieved epididymal sperm was dramatically higher than conventional IVF. Furthermore, it was at that time, in 1993, that he was able to show that testicular sperm (necessary when there is no epididymal sperm retrieval possible), also gave high fertilization and pregnancy rates using ICSI, and published these studies in Human Reproduction and in Fertility and Sterility. He even demonstrated that pregnancy rates and delivery rates using frozen/thawed retrieved epididymal sperm using ICSI were no different from fresh. Finally, he demonstrated that even in cases of non-obstructive azoospermia, where seemingly there is no sperm production, in 60% of the cases, some tiny amount of sperm can nonetheless be found in the testes which can be used for ICSI, and result in normal pregnancy and delivery. This was true whether the cause of non-obstructive azoospermia was Sertoli cell only, maturation arrest, cryptorchidism, or even post-chemotherapy azoospermia.

12. Y Chromosome Deletions

In 1995, three years after embarking on a major mapping study of the Y chromosome in his azoospermic patients, in cooperation with Dr. David Page’s lab at MIT, he was the first to report DAZ deletions associated with 13% of cases of non-obstructive azoospermia. This was the seminal study that has led to a huge proliferation of further studies on the association of Y chromosome deletions or mutations, with male infertility. In 1998, he reported that large Y chromosome deletions were associated with finding no sperm in the testes at TESE/ICSI attempts, and the smaller Y deletions, limited just to AZFc, resulted either in severe oligospermia or in a high percentage of azoospermic cases having some sperm retrievable. A year or two later, in 1999, he was the first to demonstrate that azoospermic men with infertility caused by AZFc deletions can produce sons by intracytoplasmic injection of sperm, and genetic analysis of these sons revealed that the AZFc deletion is transmitted to the male offspring, who is likely to have the same infertility problem as his father.

Finally, in 2001, the AZFc region of the Y chromosome was completely sequenced, demonstrating unprecedented structural features, including massive palindromes and uniform recurrent amplicons, which now helps to explain the genetic cause of many cases of severe male infertility. In that same year in Nature Genetics, he also published mathematical projections of the likelihood of proliferation of male infertility in future generations as a result of ICSI, assuming a genetic cause that will be transmitted from father to son. This chromosome sequencing was performed by the MIT Program, but it was a culmination of the previous nine years of studies on Y deletions in men with male infertility begun by a specific plan outlined by Dr. Silber and Dr. David Page, and ultimately leading to a gigantic amount of molecular work by many researchers. However, the seed of this whole field began in a joint collaboration with Dr. David Page as the molecular biologist and Dr. Silber as the clinician in 1992.

Dr. Silber is now spearheading a massive, long-term study in Japan with the Page lab, and with the University of Amsterdam, to determine, in more intricate detail via association studies, the genetic determinants of sperm count throughout the genome.

13. Effect of Female Age and Ovarian Reserve On Pregnancy Rate in Male Infertility, and PGD for male factor infertility

With the publication of his paper in 1997 in Human Reproduction, he was the first to show that even in the most severe cases of male infertility, the major predictors of success with IVF and retrieved sperm, are the age of the wife and her ovarian reserve. The severity of the oligospermia or even azoospermia had no major impact. It wasn’t until 2003 that he was finally able to also show a very subtle effect of the sperm in cases of the most severe spermatogenic defects, published in Fertility and Sterility. He demonstrated this via PGD comparing chromosomal abnormalities in patients with TESE for non-obstructive azoospermia versus patients undergoing ICSI with ejaculated sperm for moderate oligospermia. So although the female factors are most significant, he was able to identify cases where male factor also had an impact on pregnancy and delivery rates with ICSI.

14. Extinction of the Dinosaurs

The study of the evolution of the X and Y chromosome and how it results in a high frequency of male infertility caused him to speculate and write a detailed paper on environmental versus genetic sex determination. The continual evolution, over and over again, of specific sex determining genes or sex determining chromosomes, whether in earth worms, fruit flies, birds, or mammals, is remarkable. The sex determining gene on the Y chromosome of mammals is completely different from the sex determining gene of the Y chromosome in fruit flies. Over and over again throughout evolutionary time and in all the different genes, a genetic sex determining mechanism has independently evolved, which means that there must be some survival benefit to genetic sex determination. However, we know that genetic sex determination, for example in mammals, with the arrival of an SRY gene resulting in a Y chromosome, with all of its amplicons and palindromes and susceptibilities to deletion, is the major cause of male infertility. Therefore, he asked, “why would such a detrimental sex determining gene always develop eventually in any genus over time?”

A comparative phylogenetic study by Dr. Silber, Dr. Dave Miller and mathematician, Dr. Jonathan Summers, revealed that the benefit to this evolution is a guaranteed (usually 50:50) sex ratio regardless of extreme environmental changes. In alligators and crocodiles and turtles, and some fish, the temperature in which the embryo incubates, determines whether it will be a male or a female, related to enzyme amplification such as aromatase. However, eventually in most species, a sex determining chromosome develops which guarantees a 50:50 ratio no matter what the temperature and no matter what the environmental conditions.

They showed that dinosaurs became extinct because of their temperature-dependent sex determination system that was similar to modern day crocodiles, alligators and turtles. But unlike crocodilians, the inability of dinosaurs to migrate up and down streams to adjust the temperature of egg incubation to outside conditions, led to their extinction, not from a generally toxic environment, but to a skewed sex ratio of all males so that they could not reproduce. Birds are the only surviving dinosaur because they evolved a ZZ-ZW sex determining system 150,000,000 years ago. Thus despite the disastrous effect of global climate change on most dinosaurs, birds survived because of their evolving a genetic sex determination system. That is also why mammals, amphibians, flying insects, and even snakes survived. They also had evolved a genetic sex determination system that assured a balanced sex ratio in offspring despite global temperature change.

Crocodiles and alligators (even turtles) survived despite having a temperature based sex determination system like the dinosaurs, because of their ability to alter where they lay their eggs up and down a river bed or coast and find a temperature that would give a balanced sex ratio to their offspring.

Although this seems far removed from “reproductive” research, it was interesting that his study of the genetic origins of male infertility, helped him to discover an answer to the biggest question we have all had in our childhood, “what happened to the dinosaurs?”

15. Follow-Up of Thirty Years of Microscopic Vasectomy Reversal

In 2004, he published in the Journal of Andrology, an immense follow-up study covering thirty years of experience in over 4000 cases performed by the same surgeon for vasectomy reversal. He was able to compare the results with vasectomy reversal (involving vasoepididymostomy in more than half the cases) to recovery of fertility after other methods of contraception. Overall, there was a remarkable 89% spontaneous pregnancy rate over a long-term follow-up in couples who had either vasovasostomy or vasoepididymostomy (whichever was indicated) to reverse their vasectomy.

He published in the New England Journal of Medicine in June 2005, the first case of successful ovary transplantation between identical twin sisters discordant for ovarian function. One identical twin had undergone menopause at the age of thirteen and the other, still at age 24, had already had three children, was on contraception, and donated one of her ovaries to her sister with severe POF. He decided not to use the vascular technique which he had used in the testicular transplantation, but rather the cortical grafting technique first devised by Gosden in 1994 in sheep. In this manner, he was able to perform the operation as a simple outpatient procedure, minimally invasive with minimal risks. He carefully monitored the recovery of cycling in the recipient twin and compared that to current concepts of the physiology of resting oocytes and developing follicles. In the fourth month after the transplantation, the patient began to cycle normally and in the fifth month, she became spontaneously pregnant. The pregnancy proceeded uneventfully, and she delivered a normal baby girl via a spontaneous routine vaginal delivery. This was published immediately in the New England Journal of Medicine in June of 2005. This patient is now pregnant again with her second child, more than two years following her ovarian transplant.

Perhaps more significantly, he has now performed seven such transplants between identical twins discordant for ovarian function, with a history almost identical to that of the first pair. Thus, he actually has a series of five identical twins completely discordant for ovarian function undergoing ovarian transplantation, who have undergone ovary transplantation. The technique he developed for human ovarian transplantation is a minimally invasive outpatient procedure and is no longer surgically problematic. There are two more such cases about to be performed. All of the recipients thus far are now ovulating and menstruating, and there are three spontaneous pregnancies.

For that reason it is now possible to consider mild immunosuppression with HLA-matched patients and to go beyond monozygotic twins for this procedure. He has received approval now for transplantation between non-identical sisters for whom egg donation is forbidden by religion. More importantly, it demonstrates that the frozen cortical ovarian tissue from young cancer patients should be able to function normally when transplanted back after she is cured of her cancer, and have normal reproductive function. Furthermore, it is even possible for freezing of ovarian tissue to preserve fertility for young women who, for whatever reason, cannot have children until a much later age by which time they may have otherwise lost their fertility.

Finally, he, Dr. Roger Gosden, and Dr. Andy Feinberg, are engaged in a detailed molecular study to try to uncover the genetic causes of premature ovarian failure and the genetic determinants of ovarian reserve, using whole genome RNA expression arrays. The major reason for the huge epidemic increase in infertility in the world is the putting off of child bearing in the more complex society we live in today till about ten years later than had been routine for women of reproductive age. By studying these rare twin cases molecularly, there is an opportunity to better understand why some women are born with a large ovarian reserve and others with a deficient ovarian reserve, and thus to understand the genetic basis of the biological clock of women.

17. Gathering of anthropological and paleontological research for his new book, a culmination of 20 years of work in the field:

Y Men Are Different from Women: A history of the Y chromosome in ancient man, modern man, different societies, and in wild animals, and how it affects the behavior of men compared to women.